The essential genome of Streptococcus agalactiae.

BACKGROUND: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis. METHODS: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries-with at least 135,000 mutants per library-at serial 24 h time points after outgrowth in rich media. RESULTS: After statistical analysis of transposon insertion density and distribution, we identified 13.5 % of genes as essential and 1.2 % as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93 % concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS. CONCLUSIONS: We believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.This work was supported by NIH/NIAID R01 AI092743, R33 AI098654, and R21 AI11020 to A.J.R.; NIH/NICHD K23 HD065844 to T.M.R.; John M. Driscoll, Jr., M.D. Children’s Fund (Columbia University Department of Pediatrics) and the Pediatric Scientist Development Program (NIH/NICHD K12 HD000850) to T.A.H

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca2+ influx via voltage-gated L-type Ca2+ channels activates Ca2+ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca2+]i transients in hESC-CMs required Ca2+ entry through L-type Ca2+ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca2+ or treatment with diltiazem, an L-type channel inhibitor. Ca2+ release from the SR also contributes to the [Ca2+]i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca2+ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca2+ currents (ICa) and corresponding whole-cell [Ca2+]i transients in hESC-CMs and hFVMs, and the amplitude of both ICa and the [Ca2+]i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca2+]i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca2+ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca2+ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca2+ release by the ICa during EC coupling develops early in human cardiomyocytes

Knowledge, experience, and potential risks of dating violence among Japanese university students: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The Domestic Violence Prevention Act came into effect in Japan in 2001, but covers only marriage partner violence and post-divorce partner violence, and does not recognize intimate partner violence (IPV). The present study was performed to determine the experience of harassment, both toward and from an intimate partner, and recognition of harassment as IPV among Japanese university students.</p> <p>Methods</p> <p>A self-administered questionnaire survey regarding the experience of harassment involving an intimate partner was conducted as a cross-sectional study among freshman students in a prefectural capital city in Japan.</p> <p>Results</p> <p>A total of 274 students participated in the present study. About half of the subjects (both male and female students) had experience of at least one episode of harassment toward or had been the recipient of harassment from an intimate partner. However, the study participants did not recognize verbal harassment, controlling activities of an intimate partner, and unprotected sexual intercourse as violence. Experience of attending a lecture/seminar about domestic violence and dating violence did not contribute to appropriate help-seeking behavior.</p> <p>Conclusions</p> <p>An educational program regarding harassment and violence prevention and appropriate help-seeking behavior should be provided in early adolescence to avoid IPV among youth.</p

A closed loop brain-machine interface for epilepsy control using dorsal column electrical stimulation

Although electrical neurostimulation has been proposed as an alternative treatment for drug-resistant cases of epilepsy, current procedures such as deep brain stimulation, vagus, and trigeminal nerve stimulation are effective only in a fraction of the patients. Here we demonstrate a closed loop brain-machine interface that delivers electrical stimulation to the dorsal column (DCS) of the spinal cord to suppress epileptic seizures. Rats were implanted with cortical recording microelectrodes and spinal cord stimulating electrodes, and then injected with pentylenetetrazole to induce seizures. Seizures were detected in real time from cortical local field potentials, after which DCS was applied. This method decreased seizure episode frequency by 44% and seizure duration by 38%. We argue that the therapeutic effect of DCS is related to modulation of cortical theta waves, and propose that this closed-loop interface has the potential to become an effective and semi-invasive treatment for refractory epilepsy and other neurological disorders.We are grateful for the assistance from Jim Meloy for the design and production of the multielectrode arrays as well as setup development and maintenance, Laura Oliveira, Terry Jones, and Susan Halkiotis for administrative assistance and preparation of the manuscript. This work was funded by a grant from The Hartwell Foundation.info:eu-repo/semantics/publishedVersio

ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells

Osteosarcoma is the most common malignant bone neoplasia occurring in young patients in the first two decades of life, and represents 20% of all primitive malignant bone tumours. At present, treatment of metastatic osteosarcoma is unsatisfactory. High-dose chemotherapy followed by CD34+ leukapheresis rescue may improve these poor results. Neoplastic cells contaminating the apheresis may, however, contribute to relapse. To identify markers suitable for detecting osteosarcoma cells in aphereses we analysed the expression of bone-specific genes (Bone Sialoprotein (BSP) and Osteocalcin) and oncogenes (Met and ErbB2) in 22 patients with metastatic osteosarcoma and six healthy stem cell donors. The expression of these genes in aphereses of patients affected by metastatic osteosarcoma was assessed by RT–PCR and Southern blot analysis. Met and Osteocalcin proved to be not useful markers since they are positive in aphereses of both patients with metastatic osteosarcoma and healthy stem cell donors. On the contrary, BSP was expressed at significant levels in 85% of patients. Moreover, 18% of patients showed a strong and significantly positive (seven to 16 times higher than healthy stem cell donors) ErbB2 expression. In all positive cases, neoplastic tissue also expressed ErbB2. Our data show that ErbB2 can be a useful marker for tumour contamination in aphereses of patients affected by ErbB2-expressing osteosarcomas and that analysis of Bone Sialoprotein expression can be an alternative useful marker

First report of the ectomycorrhizal status of boletes on the Northern Yucatan Peninsula, Mexico determined using isotopic methods

Despite their prominent role for tree growth, few studies have examined the occurrence of ectomycorrhizal fungi in lowland, seasonally dry tropical forests (SDTF). Although fruiting bodies of boletes have been observed in a dry tropical forest on the Northern Yucatan Peninsula, Mexico, their occurrence is rare and their mycorrhizal status is uncertain. To determine the trophic status (mycorrhizal vs. saprotrophic) of these boletes, fruiting bodies were collected and isotopically compared to known saprotrophic fungi, foliage, and soil from the same site. Mean δ15N and δ13C values differed significantly between boletes and saprotrophic fungi, with boletes 8.0‰ enriched and 2.5‰ depleted in 15N and 13C, respectively relative to saprotrophic fungi. Foliage was depleted in 13C relative to both boletes and saprotrophic fungi. Foliar δ15N values, on the other hand, were similar to saprotrophic fungi, yet were considerably lower relative to bolete fruiting bodies. Results from this study provide the first isotopic evidence of ectomycorrhizal fungi in lowland SDTF and emphasize the need for further research to better understand the diversity and ecological importance of ectomycorrhizal fungi in these forested ecosystems